CN115820939A - CrRNA for detecting monkeypox virus, nucleic acid molecule composition, detection system and application - Google Patents
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Abstract
The invention discloses a crRNA for detecting monkeypox virus, a nucleic acid molecule composition, a detection system and application, wherein the crRNA comprises at least one group of nucleotide sequences shown as SEQ ID NO 1-6. The nucleic acid molecule composition comprises a crRNA and an RAA primer pair, wherein the crRNA comprises nucleotide sequences shown in SEQ ID NO 1-6. The detection system is a CRISPR-Cas12 detection system or a CRISPR-Cas13 detection system or a CRISPR-Cas12-Cas13 dual-channel detection system. The monkey pox virus detection kit is based on monkey pox virus genes, designs monkey pox virus specific crRNA, is combined with a CRISPR-Cas fluorescence/aptamer detection system, can detect whether monkey pox viruses exist in a short time, can specifically distinguish different subtypes of monkey pox viruses, is convenient to operate, does not need precise instruments, and has good specificity and high sensitivity, and the minimum detection limit can reach 4copies. The CRISPR-Cas12-Cas13 dual-channel detection system established by the invention can detect whether other viruses except the monkeypox virus exist in a sample, thereby further enriching the detection content.
Description
Technical Field
The invention relates to the technical field of pathogen detection. More particularly, relates to a crRNA for detecting monkeypox virus, a nucleic acid molecule composition, a CRISPR-Cas detection system and a kit.
Background
Monkeypox is a zoonosis caused by orthopoxvirus, which causes human beings to suffer from smallpox-like diseases. Early symptoms of monkey pox infection include fever, headache, muscle pain, swollen lymph nodes, chills and tiredness, with a blister-like rash on the back and healing. Enhanced monitoring and detection of monkeypox cases is an important tool in understanding the changing epidemiology of this disease.
The CRISPR-Cas system is widely present in bacteria and archaea, and constitutes an acquired immune system against foreign invader nucleic acids, and the defense process thereof comprises three stages: spacer acquisition, synthesis of crRNA and target interference. Generally, CRISPR-Cas systems consist of CRISPR RNA (crRNA) and a Cas protein, the crRNA is complementary to the target sequence and can direct the Cas protein for sequence-specific recognition and cleavage.
The Cas12 and Cas13 systems in the CRISPR-Cas system are different from the Cas9 system, cas12 and Cas13 proteins have trans-cleavage activity, and the proteins specifically target double-stranded DNA/single-stranded RNA (dsDNA/RNA) and activate the trans-cleavage activity under the guide of crRNA, and indiscriminately cleave non-specific single-stranded DNA/RNA (ssDNA/ssRNA). The Cas12 system recognizes double-stranded DNA, and recognizes single-stranded DNA through trans-cleavage; the Cas13 system recognizes RNA, which is also recognized by trans-cleavage. It was found that, at targeted RNA concentrations of 1-10 pmol/L, only 0.02% of the Cas13a system was activated, whereas the cleavage efficiency reached 25-50%, and each targeted cleavage resulted in a subsequent increase in nonspecific targeted cleavage activity, amplifying the signal, and allowing the detection of a specifically enhanced fluorescent signal in a short time. Cas12 and Cas13 systems are therefore rapidly evolving in the field of detection.
The diagnosis of the virus comprises clinical diagnosis and laboratory diagnosis, because of the complex situation of the monkeypox virus, the most effective way for confirming the monkeypox is to carry out the laboratory diagnosis at present, and the commonly used methods comprise etiology diagnosis, serodiagnosis, molecular biology diagnosis and the like. The most traditional detection method is a virus isolation method, but the method is long in time-consuming experiment period. The existing rapid detection method is a real-time fluorescence PCR method, but the probe is expensive to purchase, and a complex thermal cycle system is required to achieve the purpose of amplifying nucleic acid, so that the method cannot be applied to real-time diagnosis. Compared with the prior art, the advantages of the isothermal amplification technology are highlighted, and the isothermal amplification technology becomes a promising field detection method. The recombinase-mediated isothermal nucleic acid amplification technology (RAA) can realize the massive amplification of samples under the condition of constant temperature in a short time, has simple operation and is not limited by instruments. However, when the concentration of the template is low, non-specific amplification may occur, and false positives are likely to occur.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a crRNA and nucleic acid molecule composition for detecting the monkeypox virus, which can be combined with DNA/RNA of the monkeypox virus to detect. The second purpose of the invention is to provide a simian pox virus detection CRISPR-Cas detection system, which can detect whether the simian pox virus exists or not in a short time and can specifically distinguish different subtypes of simian pox viruses.
In order to achieve the aim, the invention discloses a crRNA for detecting monkeypox virus, which comprises at least one group of nucleotide sequences shown as SEQ ID NO 1-6.
Further, the crRNA comprises nucleotide sequences shown as SEQ ID NO 1-6.
The invention discloses a nucleic acid molecule composition for detecting monkeypox virus, which comprises a crRNA and an RAA primer pair, wherein the crRNA comprises a nucleotide sequence shown by SEQ ID NO 1-6, and the RAA primer pair comprises at least one group of the following primer pairs:
(1) The upstream primer sequence shown as SEQ ID NO 7 and the downstream primer sequence shown as SEQ ID NO 8.
(2) An upstream primer sequence shown by SEQ ID NO 9 and containing a T7 promoter and a downstream primer sequence shown by SEQ ID NO 10.
(3) The upstream primer sequence shown as SEQ ID NO 11 and the downstream primer sequence shown as SEQ ID NO 12.
(4) An upstream primer sequence shown by SEQ ID NO 13 and containing a T7 promoter and a downstream primer sequence shown by SEQ ID NO 14.
(5) The upstream primer sequence shown as SEQ ID NO 15 and the downstream primer sequence shown as SEQ ID NO 16.
(6) An upstream primer sequence shown by SEQ ID NO 17 containing a T7 promoter and a downstream primer sequence shown by SEQ ID NO 18.
The invention also discloses application of the crRNA or nucleic acid molecule composition in detection of the monkeypox virus.
Further, the invention discloses a CRISPR-Cas detection system for detecting monkeypox virus, which comprises the crRNA or the nucleic acid molecule composition.
Preferably, the detection system is a CRISPR-Cas12 detection system or a CRISPR-Cas13 detection system or a CRISPR-Cas12-Cas13 dual-channel detection system.
Preferably, the kit further comprises a ssRNA reporter molecule or/and a ssDNA reporter molecule, wherein the sequence of the ssDNA reporter molecule is 5'-FAM-TTATT-BHQ1-3' or 5'-HEX-TTATT-BHQ1-3'; the sequence of the ssRNA reporter molecule is 5 '-FAM-rUrUrUrU-BHQ 1-3'.
Preferably, the kit further comprises a ThT aptamer or a manno III aptamer, the sequence of the ThT aptamer being:
5’-CGAGGCTATTAGGAGGTGGGATGC-3’。
the sequence of the Mango III aptamer is as follows:
5’-GGCACGUACGAAGGAAGGAUUGGUAUGUGGUAUAUUCGUACGUGCC-3’。
the dual-channel detection comprises the detection of the mixed infection of the monkeypox virus and any one of the monkeypox virus subtypes, the HIV virus and the new coronavirus, or the detection of the mixed infection of the two monkeypox virus subtypes.
The invention also discloses a monkey pox virus detection kit, which comprises the crRNA, the nucleic acid molecule composition or the CRISPR-Cas detection system.
Compared with the prior art, the invention has the advantages that:
1. the detection method of the invention designs the monkey pox virus specific crRNA based on the monkey pox virus gene, and can specifically distinguish different subtypes of monkey pox viruses by combining a CRISPR-Cas fluorescence/aptamer detection system, and the detection method is convenient to operate and does not need precise instruments. The method is simple, good in specificity and high in sensitivity, the minimum detection limit can reach 4 copies/mu L, and the method is visualized under ultraviolet.
2. The CRISPR-Cas detection system for detecting the monkeypox virus is a method for detecting the monkeypox virus based on CRISPR-Cas detection combined with an RAA constant-temperature amplification technology, can detect whether the monkeypox virus exists or not in a short time, and has wide detection coverage.
3. The invention establishes a CRISPR-Cas dual-channel detection method, combines a Cas12a system and a Cas13a system to establish dual-channel detection, comprises a RAA-CRISPR-Cas12a/13a probe method, a RAA-CRISPR-Cas12a/13a combined ThT/Mango aptamer method and CRISPR-Cas12a-Cas13a double-gene detection, and provides a scheme for monitoring, controlling and preventing monkeypox and other diseases. Can enlarge the detection range of the monkey pox virus, can identify the monkey pox virus subtype, and can detect the mixed infection of the monkey pox virus and other viruses (such as HIV virus or new coronavirus) or two monkey pox virus subtypes.
Drawings
FIG. 1 is a diagram of specificity test.
FIG. 2 is a graph showing sensitivity of RAA-CRISPR-Cas12a probe method for detecting monkeypox virus.
FIG. 3 is a graph of sensitivity of RAA-CRISPR-Cas12a probe method for detecting monkeypox virus cono-branches.
FIG. 4 is a diagram of the sensitivity of RAA-CRISPR-Cas12a probe method for detecting simian pox virus West non-branching.
FIG. 5 is a graph showing sensitivity of RAA-CRISPR-Cas12a-ThT aptamer method for detecting monkeypox virus.
FIG. 6 is a graph showing the sensitivity of RAA-CRISPR-Cas13a probe method for detecting monkeypox virus.
FIG. 7 is a graph of sensitivity of RAA-CRISPR-Cas13a probe method for detecting monkeypox virus cono-branches.
FIG. 8 is a diagram of the sensitivity of RAA-CRISPR-Cas13a probe method for detecting simian pox virus west non-branching.
FIG. 9 is a graph showing sensitivity of RAA-CRISPR-Cas13a-Mango aptamer method for detecting monkeypox virus.
Fig. 10 is a CRISPR-Cas12a-13a two-channel detection diagram.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the present invention is further described in detail with reference to the specific embodiments below.
The invention discloses a monkeypox virus detection method which comprises the steps of designing specific crRNA, transcribing in vitro and purifying, expressing and purifying Cas12a/13a protein, extracting plasmids containing monkeypox virus partial fragments, establishing a CRISPR-Cas detection system, establishing an RAA-CRISPR-Cas detection method, establishing a CRISPR-Cas12a-13a double-channel detection method and judging results. The specific process is detailed below.
EXAMPLE I establishment of CRISPR-Cas detection System
S1: primer design of CRISPR-Cas-based monkeypox virus detection method
1. Design of specific crRNA
The crRNA is designed by registering NCBI and using 3360 monkeypox virus gene sequences in GenBank as a detection target region through comparison. The monkey pox virus general crRNA is selected from F3L segment, the monkey pox virus congol branch crRNA is selected from C3L segment, and the monkey pox virus West non-branch sgRNA is selected from G2R segment.
In vitro transcription and purification of crRNA
The designed 5' end of the sequence of crRNA is introduced into the T7 sequence, and a template for in vitro transcription is synthesized. crRNA was prepared by in vitro Transcription using T7RAN Transcription Kit (T7 High Yield RNA Transcription Kit, novozam). The crRNA sequences are shown in Table 1.
Table 1: sequence listing of crRNA
3. Constructing recombinant plasmids pUC57-MPXV, pUC57-MPXV-CA and pUC57-MPXV-WA containing the target sequence, downloading the gene sequence aiming at the detection of the monkeypox virus, and synthesizing the gene sequence by a general biology company. The sequence is shown in Table 2.
S2, expression and purification of LbCas12a/huLwCas13a protein in prokaryotic system
1. Protein expression
Obtaining plasmid pMBP-LbCas12a on Addgene, comprising a 10XHis-MBP tag, a TEV site; plasmid pC 013-Twintrepp-SUMO-huLwCas 13a, comprising a 6XHis tag, the SUMO site. The plasmid was taken and transformed into BL21 (DE 3) competent cells. After screening and identification, the cells were shake-cultured overnight at 37 ℃ and 180r/min in LB liquid medium containing 100. Mu.g/mL ampicillin. The next day, the bacterial solution was transferred to a new LB liquid medium containing 100. Mu.g/mL ampicillin in a ratio of 1.
2. Protein purification
The cell suspension obtained in step 1 was centrifuged at 4 ℃ and 3500rpm for 30min to collect precipitates, resuspended in buffer A (25mM Tris,150mM NaCl,25mM Imidazole,10% glycerol, final pH 7.5), then repeatedly freeze-thawed 3 times, and sonicated (sonication conditions: 3s, 2s rest, 35% power) using an ultrasonic cell disrupter (model KS-1200DN, kunshanjiemei sonicator Co., ltd.) until the cell suspension was clear. Then, the mixture was centrifuged at 20000rpm at 4 ℃ for 1 hour, and the supernatant was collected.
The supernatant was loaded onto 5mL of His TratpTM FF crud (G EHeathcare Life Sciences), and the target protein was eluted with a protein purifier (AKTAexplorer, GE Co.) and verified by SDS-PAGE, followed by digestion. The enzyme-cleaved products were loaded onto 5mL His TraptM FF crudes (G E Healthcare Life Sciences), proteins were eluted by a protein purifier, and after SDS-PAGE verification, protein concentration was determined by a BCA (Bicinchoninic Acid) protein quantification kit and stored at-80 ℃.
S3, preliminarily establishing a simian pox virus CRISPR-Cas12a/13a detection system
Screening for optimal ratio of LbCas12a to crRNA
Combining LbCas12a with crRNA according to different concentrations, the molar ratio of LbCas12a to crRNA was from 1:0.5 to 1:2,0.25 is a gradient, lbCas12 is 100nM, pUC57-Monkeypox is used as positive template, pUC57 no-load is used as negative control, and the optimal reaction system is selected under the same reaction condition. Considering the cost and the experimental effect, the final optimal reaction system is 100nM LbCas12a and 200nM crRNA.
Optimal ratio screening of huLwCas13a to crRNA
The molar ratio of huLwCas13a to crRNA is recommended to be 2 according to the literature: 1, so huLwCas13a in the late reaction system was 100nM and crRNA was 50nM.
CRISPR-Cas12a probe method
The reaction system is as follows:
(1) 100nM purified LbCas12a;
②200nM crRNA；
(3) 2. Mu.L of plasmid DNA; plasmid DNA extraction was performed according to the FastPure Plasmid Mini Kit protocol, and the concentration was measured with a NanoPhotometer-N50, and stored at-20 ℃ for future use.
(4) 250nM quenched fluorescent ss DNA reporter (ss DNA FQ-labeled reporter): the sequence is 5'-FAM-TTATT-BHQ1-3', wherein 5'-TTATT-3',5 'end is modified into FAM, and 3' end is modified into BHQ1. Or the sequence is 5'-HEX-TTATT-BHQ1-3', wherein 5'-TTATT-3',5 'end is modified into HEX, and 3' end is modified into BHQ1;
(5) RNase inhibitor (1U/. Mu.L);
mix in Cas12a reaction buffer (20mM HEPES,10mM MgCl2, pH 7.9) in a reaction volume of 20. Mu.L.
Placing an ROCHE LightCycler 96 real-time fluorescent quantitative PCR instrument, and collecting fluorescence values. The procedure was set at 37 ℃ and fluorescence was collected every 30s, 60 times.
CRISPR-Cas13a probe method
The reaction system is as follows:
(1) 100nM purified huLwCas13a;
②50nM crRNA；
(3) an RNA template;
(4) the sequence of the 300nM quenched fluorescent ssRNA reporter (ss RNA FQ-labeled reporter) is 5 '-FAM-rUrUrUrUrUrU-BHQ 1-3', wherein 5'-rUrUrUrUrU-3',5 'end is modified to FAM, and 3' end is modified to BHQ1;
(5) RNase inhibitor (1U/. Mu.L);
mix in Cas13a reaction buffer (20mM HEPES,10mM MgCl2, pH 7.9) in a reaction volume of 20. Mu.L.
After being mixed evenly, the mixture is placed in an ROCHE LightCycler 96 real-time fluorescent quantitative PCR instrument, and the fluorescence value is collected. The procedure was set at 37 ℃ and fluorescence was collected every 30s, 60 times.
5. CRISPR-Cas12a-Cas13a probe method
The complete detection system of the CRISPR-Cas12a-Cas13a probe method is as follows:
(1) 50nM purified huLwCas13a;
②25nM Cas13a-crRNA；
(3) 300nM purified LbCas12a;
④600nM Cas12a-crRNA；
(5) 500nM quenched fluorescent ssDNA reporter;
(6) 300nM quenched fluorescent ssRNA reporter;
(7) t7RNA polymerase (2.5U/. Mu.L);
⑧NTP mix(1mM)；
(9) RNase inhibitor (1U/. Mu.L);
2 μ L of DNA template in R;
this was mixed with a reaction buffer (20 mM HEPES,10mM MgCl2, pH 7.9) to give a reaction volume of 20. Mu.L.
The ROCHE LightCycler 96 real-time fluorescent quantitative PCR instrument was set up to collect fluorescence values (FAM and HEX). The program was set up to collect fluorescence 60 times every 30s at 37 ℃.
CRISPR-Cas12a-ThT aptamer method
The reaction system is as follows:
(1) 100nM purified LbCas12a;
②200nM crRNA；
(3) 1 μ M ssDNA-ThT aptamer, of sequence
CGAGGCTATTAGGAGGTGGGATGC(SEQ ID NO 13)；
(4) RNase inhibitor (1U/. Mu.L),
(5) 2. Mu.L of plasmid DNA,
mix in Cas12a reaction buffer (100mM NaCl,50mM Tris-HCl,10mM MgCl2, 100. Mu.g/mL BSA, pH 7.9) in a reaction volume of 20. Mu.L.
Mixing uniformly, and then, 35min at 37 ℃. 5mM ThT dye was added and color development was observed under a 312nm UV lamp. The solution was excited at 425nm by placing a full-wavelength multifunctional microplate reader Varioskan Flash, emission was monitored at a wavelength range of 450-650nm, and fluorescence emission intensity at 490nm was collected.
7. CRISPR-Cas13a-Man aptamer method
The reaction system is as follows:
(1) 100nM purified huLwCas13a;
②50nM crRNA；
(3) 3 μ M of a Mango III aptamer; a Mango III aptamer having the sequence
GGCACGUACGAAGGAAGGAUUGGUAUGUGGUAUAUUCGUACGUCC(SEQ ID NO 14)；
(4) 220nM TO3-3PEG-Biotin dye;
(5) an RNA template;
(6) RNase inhibitor (1U/. Mu.L);
mix in Cas13a reaction buffer (20mM HEPES,10mM MgCl2, pH 7.9) in a reaction volume of 20. Mu.L.
Mixing uniformly, and then 20min at 37 ℃. The color development was observed under a 312nm UV lamp. The solution is excited at 580nm by placing a full-wavelength multifunctional microplate reader Varioskan Flash, emission is monitored in the wavelength range of 500-800nm, and the fluorescence emission intensity at 656nm is collected.
S4, judging a detection result
1. CRISPR-Cas12a/13a probe method/CRISPR-Cas 12a-Cas13a probe method: and adding a sample to be detected into the reaction system, wherein if the sample to be detected contains the DNA/RNA of the monkeypox virus, the crRNA sequence can be combined with the target DNA/RNA, the crRNA sequence is identified by the Cas protein, the conformation of the target DNA/RNA is changed, the cleavage activity is activated, the nonspecific cleavage quenches the fluorescent reporter molecule, and the fluorescence is released. And collecting the difference value between the initial fluorescence value and the final fluorescence value, and carrying out biological statistical analysis on the experimental group and the negative control group to determine whether the difference exists, wherein the result is a positive result if the difference exists, and the result is a negative result if the difference does not exist. Wherein, the reaction product of the CRISPR-Cas12a/13a probe method can be observed in color under ultraviolet, if the reaction solution is green, the result is positive, and if the reaction solution is transparent or light green, the result is negative.
2. CRISPR-Cas12a/13a aptamer method: and adding a sample to be detected into the reaction system, wherein if the sample to be detected contains the DNA/RNA of the monkeypox virus, the crRNA sequence can be combined with the target DNA/RNA, and the crRNA sequence is identified by the Cas protein, so that the conformation of the target DNA/RNA is changed, the cleavage activity is activated, and the ThT/Mango aptamer is cleaved in a non-specific manner. Under an ultraviolet lamp, the reaction solution is in green/red color and is a negative result, and the reaction solution is transparent or relatively in light green/light red color and is a positive result; collecting the fluorescence emission intensity at 490nm/580nm, and performing biological statistical analysis on the experimental group and the negative control group to determine whether there is a difference, wherein if there is a difference, the result is positive, and if there is no difference, the result is negative.
Example II establishment of RAA-CRISPR-Cas detection method
The RAA-CRISPR-Cas detection is continued on the basis of the first embodiment, and the specific method is as follows:
aiming at the general detection method of the monkeypox virus, the templates selected by the specificity test are as follows: murine pox virus ECTV (AF 012825), vaccinia virus CPXVA (F482758), smallpox virus VARV-1 (L22579), smallpox virus VARV-2 (NC 001611), smallpox virus VARV-3 (Y16780), monkeypox virus MPXV, NC each 2 uL add to CRISPR-Cas12a/13a detection system for specificity test; the simian pox virus West non-subtype detecting method includes the following specific test selecting template: adding 2 mu L of variola virus VARV (L22579), monkeypox virus congo branch subtype MPXV-CA, monkeypox virus West non-subtype MPXV-WA and NC into a CRISPR-Cas12a/13a detection system for specific test; the monkeypox virus congo branch subtype detecting method includes the following specific test selecting template: the specificity test is carried out by adding 2 mu L of each of the ectromelia virus ECTV (AF 482758) and the monkeypox virus congo branch subtype MPXV-CA, NC into a CRISPR-Cas12a/13a detection system. The specific sequence is shown in Table 2, and the test results are shown in FIG. 1.
TABLE 2 template sequences
S5. Establishment of RAA-CRISPR-Cas detection method
According to the two ends of the position of the screened optimal crRNA sequence, corresponding RAA primers are designed as shown in the following table 3:
TABLE 3 RAA primers
RAA reaction system and conditions: and (3) buffer solution V:25 mu L of the solution; primer F (10 μ M): 2 mu L of the solution; primer R (10 μ M): 2 mu L of the solution;
magnesium acetate I: 5 mu L of the solution; mu.L of template was added with water to give 50. Mu.L. The reaction was carried out at 39 ℃ for 30min.
Adding 2 mu L of RAA product into a CRISPR-Cas12a/13a probe system, reacting for 30min at 37 ℃, and collecting fluorescence increment to judge whether the product is positive or negative.
And (3) taking 2 mu L of RAA product to react for 30min in a CRISPR-Cas12a-ThT aptamer method system at 37 ℃, and adding 5mM ThT dye to judge whether the product is positive or negative.
And (3) taking 2 mu L of the RAA product, placing the RAA product in a CRISPR-Cas13 a-logo aptamer method system, and reacting for 20min at 37 ℃ to judge whether the product is positive or negative.
1. RAA-CRISPR-Cas12a probe method
The synthesized MPXV DNA sequence (2. Mu.L) was used as a template for RAA reaction (39 ℃,30 min). The complete detection system of the RAA-CRISPR-Cas12a probe method is as follows:
(1) 100nM purified LbCas12a;
(2) 200nM crRNA, 2. Mu.L RAA product;
(3) 250nM quenched fluorescent ssDNA reporter;
(4) RNase inhibitor (1U/. Mu.L);
mix in Cas12a reaction buffer (20mM HEPES,10mM MgCl2, pH 7.9) in a reaction volume of 20. Mu.L.
Placing an ROCHE LightCycler 96 real-time fluorescent quantitative PCR instrument, and collecting fluorescence values. The procedure was set at 37 ℃ and fluorescence was collected every 30s, 60 times.
2. RAA-CRISPR-Cas12a-ThT aptamer method
The synthesized MPXV DNA sequence (2. Mu.L) was used as a template for RAA reaction (39 ℃,30 min).
The complete detection system of the RAA-CRISPR-Cas12a-ThT aptamer method is as follows:
(1) 100nM purified LbCas12a;
(2) 200nM crRNA,2 μ LRAA product;
(3) 1 μ M ThT aptamer;
(4) RNase inhibitor (1U/. Mu.L);
mix in Cas12a reaction buffer (100mM NaCl,50mM Tris-HCl,10mM MgCl2, 100. Mu.g/mL BSA, pH 7.9) in a reaction volume of 20. Mu.L.
The mixture was placed in a PCR tube (Cellpro, jiangsu, china) and reacted in a C1000Touch thermal cycler (Bio-Rad) at 37 ℃ for 35min. 5mM ThT dye was added and color development was observed under a 312nm UV lamp. The solution was excited at 425nm by placing a full-wavelength multifunctional microplate reader Varioskan Flash, emission was monitored at a wavelength range of 450-650nm, and fluorescence emission intensity at 490nm was collected.
3. RAA-CRISPR-Cas13a probe method
The synthesized MPXV DNA sequence (2. Mu.L) was used as a template for RAA reaction (39 ℃,30 min).
The complete detection system of the RAA-CRISPR-Cas13a probe method is as follows:
(1) 100nM purified huLwCas13a;
(2) 50nM crRNA, T7RNA polymerase (2.5U/. Mu.L);
③NTP mix(1mM)；
(4) RNase inhibitor (1U/. Mu.L);
(5) 2 μ LRAA product;
(6) 250nM quenched fluorescent ssRNA reporter;
mix in Cas13a reaction buffer (20mM HEPES,10mM MgCl2, pH 7.9) in a reaction volume of 20. Mu.L.
Placing an ROCHE LightCycler 96 real-time fluorescent quantitative PCR instrument, and collecting fluorescence values. The procedure was set at 37 ℃ and fluorescence was collected every 30s, 60 times.
4. RAA-CRISPR-Cas13 a-logo aptamer method
The synthesized MPXV DNA sequence (2. Mu.L) was used as a template for RAA reaction (39 ℃,30 min).
The complete detection system of the RAA-CRISPR-Cas13a-Man aptamer method is as follows:
(1) 100nM purified huLwCas13a;
②50nM crRNA；
(3) t7RNA polymerase (2.5U/. Mu.L);
④NTP mix(1mM)；
(5) RNase inhibitor (1U/. Mu.L);
(6) 2 μ LRAA product;
⑦3μM Mango III；
(8) 220nM TO3-3PEG-Biotin dye;
mix in Cas13a reaction buffer (20mM HEPES,10mM MgCl2, pH 7.9) in a reaction volume of 20. Mu.L.
The mixture was placed in a PCR tube (Cellpro, jiangsu, china) and reacted in a C1000Touch thermal cycler (Bio-Rad) at 37 ℃ for 20min. The color development was observed under a 312nm UV lamp. The solution is excited at 580nm by placing a full-wavelength multifunctional microplate reader Varioskan Flash, emission is monitored in the wavelength range of 500-800nm, and the fluorescence emission intensity at 656nm is collected.
S6.RAA-CRSPR-Cas12a/13a sensitivity test
1. Method for detecting monkeypox virus by RAA-CRISPR-Cas12a probe method
The plasmid pUC57-MPXV is used as a template to dilute the plasmid to 2X 10 3 、2×10 2 、2×25、2×10 1 、2×10 0 copies/. Mu.L. Taking 2 mu L of the mixture to be put in an RAA reaction system and carrying out 30min at the temperature of 39 ℃. And taking 4 mu L of RAA product to perform a sensitivity test in a CRISPR-Cas12a probe method system. With empty pUC57 being a negative control. The RAA-CRISPR-Cas12a probe method can detect 4copies at the lowest, and the test result is shown in figure 2.
2. Method for detecting monkeypox virus congol branch by RAA-CRISPR-Cas12a probe method
Plasmid pUC57-MPXV-CA is used as a template to carry out 10-fold gradient dilution, namely 2 x 10 4 、2×10 3 、2×10 2 、2×10 1 、2×10 0 copies/. Mu.L. Taking 2 mu L of the mixture to be put in an RAA reaction system and carrying out 30min at the temperature of 39 ℃. And taking 4 mu L of RAA product to perform a sensitivity test in a CRISPR-Cas12a probe method system. With empty pUC57 being a negative control. The RAA-CRISPR-Cas12a probe method can detect 40copies at the lowest, and the test result is shown in figure 3.
3. Method for detecting simian pox virus west non-branch by RAA-CRISPR-Cas12a probe method
Plasmid pUC57-MPXV-WA WAs used as a template, and the plasmid WAs diluted by 10-fold gradient to 2X 10 4 、2×10 3 、2×10 2 、2×10 1 、2×10 0 copies/. Mu.L. Taking 2 mu L of the mixture to be put in an RAA reaction system and carrying out 30min at the temperature of 39 ℃. And taking 4 mu L of RAA product to perform a sensitivity test in a CRISPR-Cas12a probe method system. With empty pUC57 being a negative control. The RAA-CRISPR-Cas12a probe method can detect 4000copies at the lowest, and the test result is shown in figure 4.
4. RAA-CRISPR-Cas12a-ThT aptamer method detection
Plasmid pUC57-MPXV is used as a template to carry out 10-fold gradient dilution, namely 2 x 10 3 、2×10 2 、2×2 5 、2×10 1 、2×10 0 copies/. Mu.L. mu.L of the obtained product was put into an RAA reaction system at 39 ℃ for 30min. And taking 2 mu L of RAA product to perform a sensitivity test in a CRISPR-Cas12a-ThT method system. RAA-CRISPR-Cas12a-ThT aptamer method can be minimally detectedTo 100copies, the results are shown in FIG. 5.
5. Method for detecting monkeypox virus by RAA-CRISPR-Cas13a probe method
Plasmid pUC57-MPXV is used as a template to carry out 10-fold gradient dilution, namely 2 x 10 4 、2×10 3 、2×10 2 、2×10 1 、2×10 0 copies/. Mu.L. mu.L of the obtained product was put into an RAA reaction system at 39 ℃ for 30min. And taking 2 mu L of RAA product to perform a sensitivity test in a CRISPR-Cas13 a-logo aptamer method system. Of these, empty pUC57 was used as a negative control. The RAA-CRISPR-Cas13a probe method can detect 4copies at the lowest, and the test result is shown in figure 6.
Method for detecting monkeypox virus congol branch by RAA-CRISPR-Cas13a probe method
Plasmid pUC57-MPXV-CA is used as a template, and gradient dilution of 10 times is carried out, namely 2X 10 4 、2×10 3 、2×10 2 、2×10 1 、2×10 0 copies/. Mu.L. Taking 2 mu L of the mixture to be put in an RAA reaction system and carrying out 30min at the temperature of 39 ℃. And taking 2 mu L of RAA product to perform a sensitivity test in a CRISPR-Cas13a probe method system. With empty pUC57 being a negative control. The RAA-CRISPR-Cas13a probe method can detect 4copies at the lowest, and the test result is shown in figure 7.
7. Method for detecting simian pox virus west non-branch by RAA-CRISPR-Cas13a probe method
Plasmid pUC57-MPXV-WA WAs used as a template, and the plasmid WAs diluted by 10-fold gradient to 2X 10 4 、2×10 3 、2×10 2 、2×10 1 、2×10 0 copies/. Mu.L. mu.L of the obtained product was put into an RAA reaction system at 39 ℃ for 30min. And (3) taking 4 mu L of RAA product to perform a sensitivity test in a CRISPR-Cas13a probe method system, wherein the RAA-CRISPR-Cas13a probe method can detect 4copies aiming at the west non-branching minimum of plasmid pUC57-monkeypox virus, and the test result is shown in figure 8.
8. Method for detecting monkeypox virus by RAA-CRISPR-Cas13a-Man aptamer method
Plasmid pUC57-MPXV is used as a template to carry out 10-fold gradient dilution, namely 2 x 10 4 、2×10 3 、2×10 2 、2×10 1 、2×10 0 copies/. Mu.L. Taking 2 mu L of the mixture to be put in an RAA reaction system and carrying out 30min at the temperature of 39 ℃. Taking 2 μ L of RAA product inAnd (3) carrying out a sensitivity test in a CRISPR-Cas13a-Man aptamer method system. With empty pUC57 being a negative control. The RAA-CRISPR-Cas13a-Man aptamer method can detect 4copies at the lowest, and the test result is shown in figure 9.
Example III establishment of double-channel detection method for CRISPR-Cas12a-13a
In this embodiment, on the basis of the first embodiment and the second embodiment, the Cas12a system and the Cas13a system are combined to establish the dual-channel detection, and the specific method is as follows:
1. CRISPR-Cas12a-Cas13a dual-channel detection probe method
The complete detection system of the CRISPR-Cas12a-Cas13a probe method is as follows:
(1) 50nM purified huLwCas13a;
②25nM Cas13a-crRNA；
(3) 300nM purified LbCas12a;
④600nM Cas12a-crRNA；
(5) 500nM quenched fluorescent ssDNA reporter;
(6) 300nM quenched fluorescent ssRNA reporter;
(7) t7RNA polymerase (2.5U/. Mu.L);
⑧NTP mix(1mM)；
(9) RNase inhibitor (1U/. Mu.L);
2 μ L of DNA template in R;
this was mixed with a reaction buffer (20 mM HEPES,10mM MgCl2, pH 7.9) to give a reaction volume of 20. Mu.L. The ROCHE LightCycler 96 real-time fluorescent quantitative PCR instrument was set up and fluorescence values (FAM and HEX) were collected. The program was set up to collect fluorescence 60 times every 30s at 37 ℃.
2. Monkey pox virus double-channel detection probe method
The complete detection system of the monkeypox virus double-channel detection probe method comprises the following steps:
(1) 50nM purified huLwCas13a;
(2) 25nM Cas13a-crRNA (monkeypox virus);
(3) 300nM purified LbCas12a;
(4) 600nM Cas12a-crRNA (monkey pox virus Congo fruit gold branch);
(5) 500nM quenched fluorescent ssDNA reporter;
(6) 300nM quenched fluorescent ssRNA reporter;
(7) t7RNA polymerase (2.5U/. Mu.L);
⑧NTP mix(1mM)；
(9) RNase inhibitor (1U/. Mu.L);
2 μ L of DNA template in R;
this was mixed with a reaction buffer (20 mM HEPES,10mM MgCl2, pH 7.9) to give a reaction volume of 20. Mu.L.
The ROCHE LightCycler 96 real-time fluorescent quantitative PCR instrument was set up to collect fluorescence values (FAM and HEX). The program was set up to collect 60 fluorescence every 30s at 37 ℃ and the results are shown in FIG. 10.
3. Monkey pox virus typing detection probe method
The complete detection system of the monkey pox virus typing detection probe method is as follows:
(1) 50nM purified huLwCas13a;
(2) 25nM Cas13a-crRNA (monkeypox virus west non-branched);
(3) 300nM purified LbCas12a;
(4) 600nM Cas12a-crRNA (monkey pox virus Congo fruit gold branch);
(5) 500nM quenched fluorescent ssDNA reporter;
(6) 300nM quenched fluorescent ssRNA reporter;
(7) t7RNA polymerase (2.5U/. Mu.L);
⑧NTP mix(1mM)；
(9) RNase inhibitor (1U/. Mu.L);
the mixture was mixed with a reaction buffer (20mM HEPES,10mM MgCl2, pH 7.9) in a reaction volume of 20. Mu.L.
The ROCHE LightCycler 96 real-time fluorescent quantitative PCR instrument was set up to collect fluorescence values (FAM and HEX). The procedure was set at 37 ℃ and fluorescence was collected every 30s, 60 times, and the results are shown in FIG. 10.
4. Double-channel detection method for HIV and monkeypox virus
The complete detection system of the double-channel detection method for the HIV and the monkeypox virus is as follows:
(1) 50nM purified huLwCas13a;
(2) 25nM Cas13a-crRNA (monkey pox virus);
(3) 300nM purified LbCas12a;
④600nM Cas12a-crRNA(HIV)；
(5) 500nM quenched fluorescent ssDNA reporter;
(6) 300nM quenched fluorescent ssRNA reporter;
(7) t7RNA polymerase (2.5U/. Mu.L);
⑧NTP mix(1mM)；
(9) RNase inhibitor (1U/. Mu.L);
2 μ L of DNA template in R;
the mixture was mixed with a reaction buffer (20mM HEPES,10mM MgCl2, pH 7.9) in a reaction volume of 20. Mu.L.
The ROCHE LightCycler 96 real-time fluorescent quantitative PCR instrument was set up and fluorescence values (FAM and HEX) were collected. The procedure was set at 37 ℃ and fluorescence was collected every 30s, 60 times, and the results are shown in FIG. 10.
5. Double-channel detection method for new coronavirus and monkeypox virus
The complete detection system of the new coronavirus and monkeypox virus double-channel detection method comprises the following steps:
(1) 50nM purified huLwCas13a;
(2) 25nM Cas13a-crRNA (monkey pox virus);
(3) 300nM purified LbCas12a;
(4) 600nM Cas12a-crRNA (neocoronavirus);
(5) 500nM quenched fluorescent ssDNA reporter;
(6) 300nM quenched fluorescent ssRNA reporter;
(7) t7RNA polymerase (2.5U/. Mu.L);
⑧NTP mix(1mM)；
(9) RNase inhibitor (1U/. Mu.L);
2 μ L of DNA template in R;
the mixture was mixed with a reaction buffer (20mM HEPES,10mM MgCl2, pH 7.9) in a reaction volume of 20. Mu.L.
The ROCHE LightCycler 96 real-time fluorescent quantitative PCR instrument was set up and fluorescence values (FAM and HEX) were collected. The procedure was set at 37 ℃ and fluorescence was collected every 30s, 60 times, and the results are shown in FIG. 10.
It should be noted that, in the embodiments of the present invention, the RAA-CRISPR-Cas12a/13a detection system is used to perform simian pox virus detection, and the CRISPR-Cas12a-Cas13a detection system is used to perform dual-channel detection on simian pox virus and its branches, simian pox virus and HIV, simian pox virus and new coronavirus. For those skilled in the art, reading the present invention, other CRISPR-Cas detection systems can be used to implement the inventive scheme.
Example four
The embodiment discloses a monkey pox virus detection kit, which comprises the crRNA, or the nucleic acid molecule composition, or the CRISPR-Cas detection system in the embodiment.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (10)
1. A crRNA for use in detection of monkeypox virus, characterized in that: the crRNA comprises at least one group of nucleotide sequences shown as SEQ ID NO 1-6.
2. The crRNA for monkeypox virus detection according to claim 1, characterized in that: the crRNA comprises nucleotide sequences shown as SEQ ID NO 1-6.
3. A nucleic acid molecule composition for use in monkeypox virus detection, comprising: comprises crRNA and RAA primer pairs, wherein the crRNA comprises nucleotide sequences shown in SEQ ID NO 1-6, and the RAA primer pairs comprise at least one group of the following primer pairs:
(1) An upstream primer sequence shown as SEQ ID NO 7 and a downstream primer sequence shown as SEQ ID NO 8;
(2) An upstream primer sequence containing a T7 promoter shown as SEQ ID NO 9 and a downstream primer sequence shown as SEQ ID NO 10;
(3) An upstream primer sequence shown as SEQ ID NO 11 and a downstream primer sequence shown as SEQ ID NO 12;
(4) An upstream primer sequence containing a T7 promoter shown as SEQ ID NO 13 and a downstream primer sequence shown as SEQ ID NO 14;
(5) An upstream primer sequence shown as SEQ ID NO 15 and a downstream primer sequence shown as SEQ ID NO 16;
(6) An upstream primer sequence shown by SEQ ID NO 17 containing a T7 promoter and a downstream primer sequence shown by SEQ ID NO 18.
4. Use of the crRNA of claim 1 or 2 or the nucleic acid molecule composition of claim 3 in a monkeypox virus assay.
5. A CRISPR-Cas detection system for detecting monkeypox virus is characterized in that: a composition comprising the crRNA of claim 1 or 2 or the nucleic acid molecule of claim 3.
6. The CRISPR-Cas detection system for detecting the monkeypox virus according to claim 5, wherein the detection system is a CRISPR-Cas12 detection system or a CRISPR-Cas13 detection system or a CRISPR-Cas12-Cas13 dual-channel detection system.
7. The CRISPR-Cas detection system for monkeypox virus detection according to claim 6, characterized in that: the sequence of the ssRNA reporter molecule is 5'-FAM-TTATT-BHQ1-3' or the sequence thereof is 5'-HEX-TTATT-BHQ1-3'; the sequence of the ssRNA reporter molecule is 5 '-FAM-rUrUrUrU-BHQ 1-3'.
8. The CRISPR-Cas detection system for monkeypox virus detection according to claim 6, characterized in that: also included are ThT aptamers or manno III aptamers;
the sequence of the ThT aptamer is as follows: 5'-CGAGGCTATTAGGAGGTGGGATGC-3';
the sequence of the Mango III aptamer is as follows:
5’-GGCACGUACGAAGGAAGGAUUGGUAUGUGGUAUAUUCGUACGUCC-3’。
9. the CRISPR-Cas detection system for monkeypox virus detection according to claim 6, characterized in that: the dual-channel detection comprises the detection of the mixed infection of the monkeypox virus and any one of the monkeypox virus subtypes, the HIV virus and the new coronavirus, or the detection of the mixed infection of the two monkeypox virus subtypes.
10. A detection kit for monkeypox virus is characterized in that: the detection kit comprises the crRNA of claim 1 or 2, or the nucleic acid molecule composition of claim 3, or the CRISPR-Cas detection system of any one of claims 5 to 9.
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